The overall goal of the proposed research is to determine the novel mechanisms by which the Hippo signaling pathway regulates the phenotypic modulation of vascular smooth muscle cells (SMCs). Unraveling the mechanisms involved in smooth muscle phenotypic switching is an important step towards better understanding the pathology of smooth muscle-related vascular diseases. The Hippo signaling pathway is evolutionarily conserved from Drosophila to mammals and plays a critical role in controlling organ size and tumorigenesis by regulating cell proliferation and apoptosis. In mammals, cell contact and other unknown mechanisms activate the Hippo pathway core component Mst1/2 kinases to phosphorylate and activate Lats1/2 kinases, which in turn directly phosphorylate the transcriptional regulator YAP. Phosphorylated YAP is retained in cytoplasm whereas unphosphorylated form of YAP translocates into the nucleus where it binds with various transcription factors, to regulate gene expression required for control of cell proliferation and apoptosis. Our preliminary data indicate expression of Hippo-YAP pathway components in vascular smooth muscle and a novel role of Hippo-YAP pathway in phenotypic modulation. Experiments described in this proposal will critically evaluate the hypothesis that the Hippo-YAP pathway plays an integrative role in smooth muscle phenotypic modulation. In Aim 1, first we will knock down YAP expression in a rat carotid artery balloon injury model through transduction with a YAP shRNA adenovirus to determine the role of YAP in vascular lesion formation. Then we will investigate the functional role of YAP in smooth muscle development in vivo by generating a smooth muscle-specific YAP knock-out mouse. In Aim 2, we will define the role of Hippo pathway components in regulating smooth muscle phenotypic modulation. Studies are proposed to investigate the function of Hippo pathway core components, Mst1/2 and Lats1/2 in SMC phenotypic modulation by gain- and loss-of-function assays in SMCs and determine the relative importance of YAP up-regulation versus activated Hippo pathway signaling and negative regulation of YAP during vascular injury by using rat balloon injury model. In Aim 3, we will determine the mechanism by which YAP modulates smooth muscle phenotype. Preliminary data demonstrate that YAP interaction with PY motif containing transcription factors is dispensable for its function while the interaction with TEADs is essential for YAP to abrogate smooth muscle gene expression through abolishing SRF binding to CArG box within smooth muscle gene promoters. Therefore we will determine the role of TEADs in Hippo-YAP mediated smooth muscle phenotypic modulation and its underlying mechanism by gel shift, co-IP, reporter and ChIP assays. Completion of these studies will provide new insights into the mechanisms controlling smooth muscle differentiation and phenotypic modulation and identify members of the Hippo pathway that may be appropriate therapeutic targets for ameliorating vascular diseases.